A reference material for human gene molecular diagnosis, its preparation method and application

Abstract

This application relates to a molecular diagnostic reference for human gene detection, its preparation method, and its application. The reference is a recombinant cell, and the chromosome of the recombinant cell integrates the exogenous human variant gene to be detected in the form of homologous recombination; wherein the recombinant cell is selected from either recombinant mammalian cells or recombinant insect cells. This reference is substantially similar to clinical samples or cell lines of the human variant gene to be detected in terms of structure, performance, and composition, and can fully simulate gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene breakage, SNP, and gene overexpression. Furthermore, the preparation method of the reference is simple to operate, has a short preparation cycle, produces large quantities, has low production costs, and its source and performance are sufficiently stable. During use, the entire process from sample processing to detection can be monitored, exhibiting high biosafety and making it well-suited for application in molecular diagnostic products for human gene detection.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of genetic engineering, in particular to a molecular diagnostic reference for human gene detection and a preparation method and application thereof. BACKGROUND

[0002] Traditional disease diagnosis methods are phenotype diagnosis based on the phenotype of diseases or pathogens. Since the phenotype of a disease changes late, phenotype diagnosis has the disadvantages of time consumption, low precision, and even the change of some phenotypes is not specific, which can easily miss the best period of treatment. With the mapping of the human genome, the research on human genes has entered the post-genomic era. As an important milestone in the history of human exploration of their own secrets, it can help people understand their genetic information, identify the cause or predict the risk of a certain disease in the body through individual gene detection.

[0003] Gene detection is a technology for detecting DNA through blood, other body fluids or cells, and detecting DNA molecular information in the cells of the detected person after amplifying gene information. Gene detection can diagnose diseases and predict disease risks. Disease diagnosis uses gene detection technology to detect gene variations that cause genetic diseases, and predictive gene detection uses gene detection technology to find the risk of disease before the disease occurs, and takes effective preventive or intervention measures.

[0004] The most widely used gene detection at present is the detection of genetic diseases in newborns, the diagnosis of genetic diseases and the auxiliary diagnosis of some common diseases. At present, more than 1000 genetic diseases can be diagnosed by gene detection technology. Human diseases can be roughly divided into three categories: classic monogenic diseases (caused by a variation at a single gene locus), polygenic diseases (such as hypertension, diabetes and tumors) and acquired genetic diseases (i.e. infectious diseases caused by pathogenic microorganisms). Compared with traditional phenotype diagnosis methods, gene detection technology has the characteristics of high specificity, high accuracy, high sensitivity and high throughput, and is suitable for preventive detection of diseases in all populations.

[0005] The market size of the gene detection industry in China is expected to increase to 23.1 billion yuan in 2022. In the Chinese market, gene detection technologies mainly include PCR technology, gene sequencing technology, FISH technology and gene chip technology. In recent years, with the increasing application of gene detection technology in medical testing, more and more gene detection and diagnosis products have been approved, and more and more downstream enterprises have entered the research and development and production of diagnostic instruments and diagnostic kits.

[0006] In vitro diagnostic reagents refer to in vitro diagnostic reagents managed as medical devices, including reagents, kits, calibrators, quality control products and other products used for in vitro detection of human samples in the process of disease prediction, prevention, diagnosis, treatment, monitoring, prognosis observation and health status evaluation. Human gene detection related reagent evaluation national reference material refers to the reference material for quality evaluation of human gene detection related reagents, the raw materials of which should be consistent or close to clinical samples as much as possible, mainly derived from clinical samples, immortalized cell lines, gene edited cell lines, and also from genetically engineered products and recombinant plasmids, etc., should have sufficient stability, and be verified by internationally recognized methods, and as far as possible to simulate real samples, such as plasmid as reference sample needs to study the matrix effect of sample matrix. Human gene detection related reagent evaluation national reference material is usually composed of positive reference sample, negative reference sample, detection limit reference sample and precision reference sample, which is used to control the quality of diagnostic reagents, mainly control the negative or positive coincidence rate of reagents, detection limit and repeatability, etc.

[0007] However, in some gene detection reagent projects, there is no national reference material and international reference material, and most products have the problem of no corresponding reference method and standard product cannot be obtained. Therefore, in the absence of national quality control products and standard products, how do in vitro diagnostic reagent enterprises design, research and develop, prepare and manage enterprise reference materials according to the characteristics of products and the purpose of clinical use, and apply them to the quality control of the company's products? Especially when detecting some rare genotypes, how to establish suitable reference materials that can fully simulate human cells for human gene detection?

[0008] At present, the quality control products of commercialized gene detection kits mainly include clinical samples, cell lines, recombinant engineering products and recombinant plasmids, but these types of quality control products have advantages and disadvantages: (1) Clinical samples are the best reference materials, which contain the required gene sequence and can evaluate the entire analysis process, but their sources are limited and may have heterogeneity. (2) Cell lines are the second best reference materials, which can be continuously prepared, have reliable sources, can be used to simulate real clinical samples and have good stability. Their importance is self-evident, but the problem of cell quality identification has been ignored by many researchers, and cell contamination and misjudgment of cell identity are very common. (3) Genomic DNA as an artificial sample has uniformity, reproducibility and known variation sites, but it is not "real" and cannot monitor the extraction process, and may also have gene rearrangement or loss. (4) Plasmid as an artificial sample has a clear gene sequence, is easy to produce, cheap, non-infectious and has good stability, but it is a non-real sample and cannot monitor the extraction process. Therefore, there is an urgent need for an artificial reference material that can be easily obtained, can be obtained in large quantities, and is closer to human cells, so as to accurately simulate it. SUMMARY

[0009] The present application aims at the deficiencies of the prior art, and provides a molecular diagnostic reference for human gene detection, which is basically close to the clinical sample or cell line of the human variant gene to be detected in structure, performance, composition and the like, can fully simulate gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene breakage, SNP and gene overexpression and the like, and the preparation method of the reference is simple in operation, short in preparation period, large in generated quantity, low in production cost, stable in source and performance, more accurate in making, can monitor the whole process of the sample from processing to detection when used, and most importantly, can fully simulate human gene deletion, gene insertion, SNP, and also has high biological safety.

[0010] Therefore, the present application provides a molecular diagnostic reference for human gene detection in the first aspect, the reference is a recombinant cell, and a human variant gene to be detected is integrated in the chromosome of the recombinant cell in the form of homologous recombination; wherein the recombinant cell is selected from any one of a recombinant mammalian cell and a recombinant insect cell.

[0011] In the present application, the recombinant cell is preferably a recombinant mammalian cell, further preferably a recombinant human cell, and more preferably a recombinant HEK293 cell.

[0012] The HEK293 cell line is an immortalized cell of a primary human embryonic kidney cell transfected with DNA of adenovirus type 5 (Ad5), expresses the transfected gene of adenovirus 5, is humanized, and is similar to the HEK293 model of adrenal cells through a series of experimental data analysis of cell genome and transcriptome, has various neuronal characteristics, and is an in vitro model of adrenal cells. The HEK293 cell not only has the ability to generate protein glycosylation profiles and establish regulatory records, but also has various advantages, and is a particularly attractive recombinant protein expression system. First, it is carboxylated on glutamic acid and sulfated on tyrosine; second, it is simple to operate, and can quickly produce a recombinant protein through transient gene expression; third, it can be used for stable recombinant protein production. So far, the HEK293 cell and its derivatives have become one of the preferred cell lines for transient or stable protein expression, are widely used in signal transduction and protein interaction research of virus packaging, and are used for rapid small-scale protein expression and biopharmaceutical production, and have become a widely used mammalian heterologous expression system for producing recombinant proteins. In view of the many advantages of the HEK293 cell line, it is feasible to insert a human variant gene into the chromosome of the HEK293 cell to construct a stable reference.

[0013] Based on the above, the exogenous human variant gene is integrated into the chromosome of the recombinant cell (such as HEK293 cell) by homologous recombination through genetic engineering, and a molecular diagnostic reference for human gene detection is first reported. The reference can fully simulate human cells, is close to clinical samples, can monitor the whole process from sample processing to detection, and has high safety, and is an ideal reference for molecular diagnostic products.

[0014] In some embodiments, the exogenous human variant gene to be detected is stably inherited with the chromosome of the recombinant cell.

[0015] In the present application, since the exogenous human variant gene to be detected can be stably inherited with the chromosome of the recombinant cell, the reference is basically close to the clinical sample or cell line of the human gene to be detected in various aspects such as structure, performance and composition, can fully simulate gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene breakage, SNP and gene overexpression, and can monitor the whole process from sample processing to detection when used, which is superior to the commonly used genomic DNA, plasmid and the like as reference.

[0016] In some embodiments, the copy number of the exogenous human variant gene to be detected in the recombinant cell is single copy or multiple copies.

[0017] In the present application, when the exogenous human variant gene to be detected is introduced into the recombinant cell, the copy number of the exogenous human variant gene in the positive recombinant cell obtained can be single copy or multiple copies. Those skilled in the art can make routine selection according to the copy number of the corresponding human wild-type gene in the human genome or the actual use. Generally, the copy number of the human variant gene to be detected in the recombinant cell is the same as the copy number of the corresponding human wild-type gene in the human genome, and since the copy number of the corresponding human wild-type gene of the human variant gene to be detected in the human genome is mostly single copy, the copy number of the exogenous human variant gene to be detected in the recombinant cell is also generally single copy. However, the copy number of the human variant gene to be detected in the recombinant cell can also be different from the copy number of the corresponding human wild-type gene in the human genome, and those skilled in the art can make selection according to the actual application.

[0018] In some embodiments, the genetic variant form in the human variant gene is selected from at least one of gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene breakage, SNP and gene overexpression.

[0019] In the present application, the genetic variation forms in the human variant gene encompass the basic forms of human genetic variations: gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene break, SNP and gene overexpression, etc., so as to fully simulate human gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene break, SNP and gene overexpression, etc.

[0020] In the present application, the exogenous human variant gene is a single variant gene or a plurality of different variant genes; when the exogenous human variant gene is a plurality of different variant genes, the plurality of different variant genes are connected in series and integrated into the same recombinant cell chromosome, or each variant gene in the plurality of different variant genes is integrated into a different recombinant cell chromosome.

[0021] The second aspect of the present application provides a preparation method of the molecular diagnostic reference product according to the first aspect of the present application, which comprises the following steps:

[0022] S1, knocking out the non-variant gene fragment corresponding to the exogenous human variant gene to be homologously recombined on the chromosome of the recombinant cell to be recombined, to obtain the recombinant cell to be recombined with the non-variant gene fragment knocked out on the chromosome;

[0023] S2, introducing a vector containing the exogenous human variant gene into the recombinant cell to be recombined with the non-variant gene fragment knocked out on the chromosome, to obtain a recombinant cell; the exogenous human variant gene is integrated in the form of homologous recombination on the chromosome of the recombinant cell.

[0024] In the present application, the knockout method of the gene fragment on the chromosome of the recombinant cell (such as HEK293 cell) and the preparation method of the vector containing the exogenous human variant gene are conventional methods in the art, and those skilled in the art can use existing conventional methods to knockout the gene fragment and prepare the corresponding vector. In some specific embodiments, the CRISPR / Cas9 gene editing system can be used to knockout the corresponding gene fragment on the chromosome of the recombinant cell (such as HEK293 cell). Meanwhile, the method of introducing the vector containing the exogenous human variant gene into the recombinant cell (such as HEK293 cell) is also a conventional method in the art, for example, liposome transfection method can be used.

[0025] In some embodiments, the method further comprises the following steps:

[0026] S3, detecting the insertion copy number of the exogenous human variant gene in the genome of the recombinant cell;

[0027] S4, calibrating the clinical sample or cell line of the human variant gene with the recombinant cell whose insertion copy number of the exogenous human variant gene has been determined, and calibrating the reference.

[0028] In some specific embodiments, in step S3, the detection method comprises the following steps:

[0029] T1, performing fluorescent quantitative PCR detection on the genomic DNA of the recombinant cell, and calculating the ratio of the starting template copy number of the exogenous human variant gene to that of the internal reference gene;

[0030] T2, multiplying the ratio by the copy number of the internal reference gene to obtain the insertion copy number of the exogenous human variant gene in the genome of the recombinant cell.

[0031] In this application, the copy number of the internal reference gene refers to the copy number of the internal reference gene in the genome of the recombinant cell. The copy number of the selected internal reference gene is generally single copy, at which time the ratio of the starting template copy number of the exogenous human variant gene to that of the internal reference gene is the insertion copy number of the exogenous human variant gene in the genome of the recombinant cell; if the ratio is about 1, it indicates that the insertion copy number of the exogenous human variant gene in the genome of the recombinant cell is single copy.

[0032] In this application, the "clinical sample or cell line of the human variant gene" refers to a reliable reference for molecular diagnosis of the human variant gene to be detected.

[0033] In some embodiments, the calibration is the consistency of the content of the human variant gene in a certain number of the recombinant cells and a certain number of the clinical sample or cell line of the human variant gene determined by the gold standard.

[0034] In this application, the clinical sample or cell line of the human variant gene determined by the gold standard (such as cell counting method) is calibrated with the recombinant cell, which can accurately calibrate the reference, and thus the accuracy of the reference for human gene detection is higher.

[0035] In this application, the recombinant cell can be accurately counted, and the cell counting method of the human variant gene clinical sample or cell line as the gold standard can be accurately counted. By comparing the consistency of the content of the human variant gene to be detected in the clinical sample or cell line and the recombinant cell containing the same number (concentration), the recombinant cell can be calibrated, and thus the molecular diagnostic reference can be accurately calibrated.

[0036] In the present application, the preparation method of the reference product is simple in operation, large in generation amount, and low in production cost. The prepared reference product is stable in source and performance, accurate in making, and has high biological safety. Meanwhile, the prepared reference product is basically close to the corresponding clinical sample or cell line, can monitor the whole process from sample processing to detection, and most importantly, can fully simulate human gene variation (gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene breakage, SNP and gene overexpression, etc.), and has high biological safety and other characteristics.

[0037] The third aspect of the present application provides an application of the molecular diagnostic reference product as described in the first aspect of the present application or the molecular diagnostic reference product prepared by the method as described in the second aspect of the present application in the preparation of a molecular diagnostic product for detecting human genes.

[0038] The present application first creatively proposes a molecular diagnostic reference product for human gene detection. The reference product is basically close to the clinical sample or cell line of the human gene to be detected in structure, performance and composition, can fully simulate gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene breakage, SNP and gene overexpression, etc., can monitor the whole process from sample processing to detection, and therefore can be better applied to the molecular diagnostic product for detecting human genes.

[0039] In the present application, the reference product can also be used as a quality control product, a standard product, a control product and a calibration product, etc. according to the specific role in the subsequent molecular diagnostic product.

[0040] In the present application, the molecular diagnostic product for detecting human genes may, for example, be a human gene extraction kit in a sample or a human gene detection kit in a sample.

[0041] In summary, the beneficial technical effects of the present application include:

[0042] 1. The reference product constructed in the present application has the advantages of simple operation, large generation amount and low production cost;

[0043] 2. The reference product constructed in the present application is stable in source and performance, accurate in making, and also has high biological safety;

[0044] 3. The reference product constructed in the present application can fully simulate different human genes (gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene breakage, SNP and gene overexpression, etc.), and when used as a reference product, it can monitor the whole process from sample processing to detection, is more easily obtained than the currently commonly used specific cell line genomic DNA, and is more comparable than the plasmid;

[0045] 4, The reference product constructed in the application is used for benchmarking with the corresponding human gene variation clinical samples or cell lines determined as the gold standard (cell counting method), and the reference product is accurately calibrated, has higher accuracy, and can become a new molecular diagnostic reference product for human gene detection. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 is a Ct value result graph of the internal reference gene β-globin and the target gene EGFR Exon 19 (E746-A750 site) amplification in three cells; wherein Figure 1 A is the internal reference gene β-globin amplification Ct value in three cells, Figure 1 B is the target gene EGFR Exon 19 (E746-A750 site) amplification Ct value in three cells. Note: Ct value 45 in the figure means that the qPCR result has no value.

[0047] Figure 2 is a Ct value result graph of the internal reference gene β-globin and the target gene EGFR Exon 20 INS (D770-N771 site insertion) amplification in three cells; wherein Figure 2 A is the internal reference gene β-globin amplification Ct value in three cells, Figure 2 B is the target gene EGFR Exon 20 INS (D770-N771 site insertion) amplification Ct value in three cells. Note: Ct value 45 in the figure means that the qPCR result has no value.

[0048] Figure 3 is a Ct value result graph of the internal reference gene β-globin and the target gene EGFR Exon 21 mutation (L858R site mutation) amplification in three cells; wherein Figure 3 A is the internal reference gene β-globin amplification Ct value in three cells, Figure 3 B is the target gene EGFR Exon 21 mutation (L858R site mutation) amplification Ct value in three cells. Note: Ct value 45 in the figure means that the qPCR result has no value. DETAILED DESCRIPTION

[0049] In order to make the application easier to understand, the application will be further described in detail below in combination with examples, which only serve an illustrative purpose and are not limited to the application range of the application. The raw materials or components used in the application can be prepared by commercial means or conventional methods if not specifically stated.

[0050] Human EGFR gene variations associated with cancer mainly occur in exon EGFR 19 deletion (19Del), exon 20 insertion (20Ins) mutation and exon 21 mutation (L858R). Therefore, the following examples constructed a stable transfection HEK293 cell line containing EGFR Exon 19-Exon 21 mutations as a reference for molecular diagnostics. The reference contains EGFR 19 deletion (19Del), exon 20 insertion (20Ins mutation) and exon 21 mutation (L858R).

[0051] Example 1: Construction of molecular diagnostic reference for detecting gene deletion

[0052] 1. Construction and calibration of reference

[0053] 1.1 Knockout of EGFR Exon 19-Exon 21 region in HEK293 cells using CRISPR / Cas9 gene editing system

[0054] ① Use the sgRNA online design website of CRISPR, http: / / crispr.mit.edu / , to design and synthesize four different sgRNAs 1-4 targeting the EGFR Exon 19-Exon 21 region gene; ② Connect the synthesized sgRNAs 1-4 with the pU6 vector skeleton region to obtain the expression vector of pU6-sgRNA 1-4; ③ Mix 3 μg of sgRNA expression vector and control vector with 6 μg of Cas9 expression vector using LipofectAMINE TM4. Liposome transfection was performed to co-transfect HEK293 cells. Cells were collected 72 hours after transfection, and sgRNA activity was detected after genomic DNA extraction. 5. The two most active sgRNAs were tandemly selected to construct the double sgRNA expression vector pCMV-hCas9-U6-double sgRNA to improve targeting efficiency. 6. Using HEK293 cell genomic DNA as a template, the upstream and downstream homologous arms of the EGFR Exon19-Exon21 gene donor DNA were amplified by PCR. Two rounds of nested PCR were performed to obtain the PCR products of the upstream and downstream homologous arms. 7. The PCR products were purified by gel electrophoresis, ligated into a T vector, and cloned for sequencing. 8. Plasmids with correct sequencing results were ligated into the pAd5-E1 shuttle vector containing EGFP and the selection gene to obtain EGFR. ⑧ The expression vector carrying two highly active sgRNAs and the EGFRExon19-Exon21 targeting donor vector plasmid were co-transfected into HEK293 cells. After 48 hours of transfection, G418 and GCV were used for two rounds of rigorous positive and negative selection and limiting dilution to obtain single-clone positive cell lines. ⑨ The targeting fragment was amplified using outer and inner nest PCR primers, and gene knockout single-clone cells were screened after sequencing. ⑩ The mRNA level of cells after knocking out the EGFRExon19-Exon21 gene was further detected by real-time fluorescence PCR to obtain HEK293 cells with stable EGFR Exon19-Exon21 knockout.

[0055] 1.2 Construction of a stable HEK293 cell line expressing EGFR Exon19-Exon21 variant

[0056] ① An in vitro synthesis of the EGFR Exon19-Exon21 variant gene fragment, which contains EGFR Exon19Del (E746-A750 sites), EGFR Exon 20INS (D770-N771 site insertion), and EGFR Exon21 mutation (L858R site mutation); ② The synthesized EGFR Exon19-Exon21 variant gene fragment was ligated with pcDNA3.1 to construct a recombinant eukaryotic expression vector; ③ The expression was performed using liposome LipofectAMINE. TM④ HEK293 cells stably knocked out EGFR Exon19-Exon21 were transfected with a recombinant eukaryotic expression vector plasmid mediated by 2000; ⑤ Two rounds of pressure selection were performed using G418 PCR 48 h after transfection; ⑥ Single-clone positive cell lines were obtained using limiting dilution; ⑦ Primers were designed and synthesized based on the EGFR Exon19-Exon21 mutant gene sequence, and the positive recombinant HEK293 cell lines selected by G418 were preliminarily identified by PCR; ⑧ Further, the insertion copy number of the target gene in the recombinant HEK293 cell genome was determined by qPCR (the ratio of the target gene to the starting template copy number of the internal reference gene was used as the insertion copy number of the target gene in the recombinant HEK293 genome; the selected internal reference gene was β-globin, and the copy number of the internal reference gene in the recombinant HEK293 cells was single copy). The stable HEK293 cell line expressing the EGFR Exon19-Exon21 mutant with single copy insertion (named EGFR-recombinant cells) was selected as a reference for the next step of the experiment.

[0057] 1.3 EGFR-recombinant cells and HEK293 cell labeling

[0058] After passage of EGFR-recombinant cells and HEK293 cells, when the cell viability was >90% and the cells were in the logarithmic growth phase, microscopic counting was performed to determine the concentration of EGFR-recombinant cells and HEK293 cells.

[0059] 2. Culture and labeling of human non-small cell lung cancer cells HCC827

[0060] ① Remove HCC827 cells (EGFR Exon 19 (E746-A750 deletion)) from liquid nitrogen and quickly immerse them in a 37℃ water bath, dissolving them completely within 1 minute; ② In a clean bench, add the dissolved cell suspension to a centrifuge tube containing 9 mL of complete culture medium, centrifuge at 1000-1200 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 1-2 mL of RPMI-1640 complete culture medium (90% RPMI-1640 + 10% FBS); ③ Add the cell suspension to a T25 flask containing 5-6 mL of complete culture medium and incubate in an incubator; ④ When the cell viability is >90% and the cells are growing stably (approximately 3-4 weeks after transfection), perform trypsin digestion and passage the cells; ⑤ When the viability of the passaged cells is >90% and they are in the logarithmic growth phase, perform microscopic counting to determine the cell concentration.

[0061] 3. qPCR detection benchmark

[0062] The labeled EGFR-recombinant cells, HEK293 cells, and HCC827 cells were serially diluted with PBS, and 10 were taken as samples. 5cells / mL, 10 3 Genomic DNA was extracted at a concentration of 1000 cells / mL. Specific primers and probes for amplifying the internal reference gene β-globin and the target gene EGFR Exon 19 (targeting the E746-A750 locus) were designed and synthesized. Ct values ​​were compared among the three cell types using qPCR. Data were analyzed using SPSS software (version 11.5). At the same concentration, the variance analysis (ANOVA) of Ct values ​​among different cell types was performed using Duncan's Multiple Range Test (DMRT) combined with the Least Significant Difference (LSD) method to evaluate the significance of differences between the mean values ​​of different cell types (P = 0.05). The amplified Ct values ​​of the internal reference gene β-globin and the target gene EGFR Exon 19 (E746-A750 locus) in the three cell types are shown below. Figure 1 As shown; where Figure 1 A represents the Ct value of the β-globin amplification of the internal reference gene in the three cell types. Figure 1 B represents the Ct values ​​of the target gene EGFR Exon 19 (E746-A750 sites) amplification in three cell types. Values ​​represent the mean ± standard deviation; identical letters indicate P > 0.05.

[0063] from Figure 1 It was found that at any given concentration, there was no significant difference in the Ct values ​​of the internal reference gene β-globin among the three cell types (P>0.05). However, the amplification of the target gene EGFR Exon 19 (E746-A750 site) was only detectable in HEK293 cells under different concentration conditions because the target gene was lost in both EGFR-recombinant cells and HCC827 cells. This confirms that the reference sample prepared by this method can adequately simulate human gene deletion samples.

[0064] Example 2: Construction of a molecular diagnostic reference for detecting gene insertion

[0065] 1. Construction and calibration of reference materials

[0066] The reference material was the EGFR-recombinant cells from Example 1 (containing EGFR Exon 20INS (D770-N771 site insertion)), and the construction and labeling were the same as in Example 1.

[0067] 2. Cultivation and labeling of EGFR D770_N771insSVD / BaF3

[0068] The EGFR D770_N771insSVD / BaF3 cells (named EGFR-ins20, with EGFR Exon 20INS (D770-N771 site insertion)) were cultured and labeled in the same way as the HCC827 cells in Example 1.

[0069] 3. qPCR detection benchmark

[0070] The labeled EGFR-recombinant cells, HEK293 cells, and EGFR-ins20 were serially diluted with PBS, and 10 were taken as samples. 5 cells / mL, 10 3 Genomic DNA was extracted at a concentration of 1000 cells / mL. Specific primers and probes for amplifying the internal reference gene β-globin and the target gene EGFR Exon 20 (targeting the D770-N771 site) were designed and synthesized. The Ct values ​​of the three cell types were compared using qPCR. Data were analyzed using SPSS software (version 11.5). For the same concentration, the variance analysis (ANOVA) of Ct values ​​in different cells was performed using Duncan's Multiple Range Test (DMRT) combined with the Least Significant Difference (LSD) method to evaluate the significance of differences between the mean values ​​of different cells (P = 0.05). The amplified Ct values ​​of the internal reference gene β-globin and the target gene EGFR Exon 20INS (inserted at the D770-N771 site) in the three cell types are shown in Figure 2. Figure 2 A represents the Ct value of the β-globin amplification of the internal reference gene in the three cell types. Figure 2 B represents the Ct values ​​for the amplification of the target gene EGFR Exon 20INS (D770-N771 insertion) in the three cell types. Values ​​represent the mean ± standard deviation; identical letters indicate P > 0.05.

[0071] from Figure 2 It was found that at any given concentration, there was no significant difference in the Ct values ​​of the internal reference gene β-globin among the three cell types (P>0.05). However, regarding the amplification of the target gene EGFR Exon 20INS (insertion at D770-N771 sites), since HEK293 cells did not undergo this target gene insertion, the target gene could not be detected in HEK293 cells under different cell concentrations. It was only detected in EGFR-recombinant cells and EGFR-ins20 cells, and the Ct values ​​of the target gene in these two cell types showed no significant difference (P>0.05). This confirms that the reference sample prepared by this method can adequately simulate human gene insertion samples.

[0072] Example 3: Constructing a reference for SNP detection

[0073] 1. Construction and calibration of reference materials

[0074] The reference material was the EGFR-recombinant cells from Example 1 (containing the EGFR Exon21 mutation (L858R site mutation)), and the construction and labeling were the same as in Example 1.

[0075] 2. Cultivation and calibration of BaF3-EGFR-L858R-L718V-Cell-Line

[0076] BaF3-EGFR-L858R-L718V-Cell-Line cells (named EGFR-SNP, with EGFR Exon21 mutation (L858R site mutation)) were cultured and labeled in the same way as HCC827 cells in Example 1.

[0077] 3. qPCR detection benchmark

[0078] The labeled EGFR-recombinant cells, HEK293 cells, and EGFR-SNPs were serially diluted with PBS, and 10 were taken as samples. 5 cells / mL, 10 3 Genomic DNA was extracted at a concentration of [number] cells / mL. Specific primers and probes for amplifying the internal reference gene β-globin and the target gene EGFR Exon21 (targeting the L858R site) were designed and synthesized. ARMS-PCR was used for EGFR L858R. Ct values ​​were compared among the three cell types using qPCR. Data were analyzed using SPSS software (version 11.5). Analysis of variance (ANOVA) for Ct values ​​at the same concentration was performed using Duncan's Multiple Range Test (DMRT) combined with the Least Significant Difference (LSD) method to evaluate the significance of differences between the mean values ​​of different cell types (P = 0.05). The amplified Ct values ​​of the internal reference gene β-globin and the target gene EGFR Exon21 mutation (L858R site mutation) in the three cell types are shown below. Figure 3 As shown; where Figure 3 A represents the Ct value of the amplification of the internal reference gene β-globin in the three cell types. Figure 3 B represents the Ct values ​​for amplification of the target gene EGFR Exon21 mutation (L858R site mutation) in three cell types. Values ​​represent the mean ± standard deviation; identical letters indicate P > 0.05.

[0079] from Figure 3It was found that at any given concentration, there was no significant difference in the Ct values ​​of the internal reference gene β-globin among the three cell types (P>0.05). However, for the amplification of the target gene EGFR Exon21 (L858R mutation), since there was no mutation in HEK293 cells, the target gene could not be detected in HEK293 cells under different cell concentrations. It could only be detected in EGFR-recombinant cells and EGFR-SNP cells, and the Ct values ​​of the target gene in these two cell types showed no significant difference (P>0.05). This confirms that the reference sample prepared by this method can adequately simulate human gene SNP samples.

[0080] It should be noted that the embodiments described above are only for explaining this application and do not constitute any limitation on this application. This application has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to this application within the scope of the claims, and revisions can be made to the invention without departing from the scope and spirit of this application. Although the application described herein relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein; on the contrary, this application can be extended to all other methods and applications with the same function.

Claims

1. A molecular diagnostic reference material for human genetic testing, characterized in that, The reference product is a recombinant cell, and a human exogenous variant gene to be detected is integrated into the chromosome of the recombinant cell in the form of homologous recombination; wherein the recombinant cell is a recombinant human cell; The preparation method of the molecular diagnostic reference product comprises the following steps: S1, knocking out a non-variant gene fragment corresponding to an exogenous human variant gene to be recombined on the chromosome of a cell to be recombined, to obtain a cell to be recombined in which the non-variant gene fragment on the chromosome has been knocked out; S2, introducing a vector containing the exogenous human variant gene into the cell to be recombined in which the non-variant gene fragment on the chromosome has been knocked out, to obtain a recombinant cell; the chromosome of the recombinant cell has the exogenous human variant gene integrated therein in the form of homologous recombination, and the exogenous human variant gene to be detected is stably inherited along with the chromosome of the recombinant cell; S3, detecting the insertion copy number of the exogenous human variant gene in the genome of the recombinant cell; S4, calibrating a clinical sample or cell line of the human variant gene against the recombinant cell in which the insertion copy number of the exogenous human variant gene has been determined, and then calibrating the molecular diagnostic reference product; The calibration is to detect the consistency of the content of the human variant gene in a certain number of the recombinant cells and a certain number of the clinical sample or cell line of the human variant gene determined by a gold standard, by using a qPCR method or a ddPCR method.

2. The molecular diagnostic reference according to claim 1, characterized in that, The copy number of the exogenous human variant gene to be detected in the recombinant cell is single copy or multiple copies.

3. The molecular diagnostic reference according to claim 1 or 2, characterized in that, The genetic variant form in the human variant gene is selected from at least one of gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene breakage, SNP, and gene overexpression.

4. The molecular diagnostic reference according to claim 1 or 2, characterized in that, The exogenous human variant gene is a single variant gene or multiple different variant genes; when the exogenous human variant gene is multiple different variant genes, the multiple different variant genes are connected in series and integrated into the same recombinant cell chromosome, or each of the multiple different variant genes is integrated into a different recombinant cell chromosome.

5. Use of the molecular diagnostic reference product according to any one of claims 1-4 in the preparation of a molecular diagnostic product for detecting a human gene.

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